Over the years many techniques for the genetic transformation of higher organisms (animals and plants) have been developed. In these techniques it is the ultimate goal to obtain a transgenic organism, e.g. a plant, in which all cells contain a foreign DNA comprising a gene of interest (the so-called transgene) stably integrated in their genome, particularly their nuclear genome.
Transformation is a complex process which always involves the contacting of starting cells with a DNA, usually a DNA comprising foreign gene(s) of interest. The contacting of the cells with the DNA is carried out under conditions that promote the uptake of the DNA by the cells and the integration of the DNA, including the gene(s) of interest into the genome of the cell.
Starting cells for transformation are usually cells that have been cultured in vitro for some time. After contacting the cells with the DNA, the transformed cells generally need to be cultured in vitro for a certain period in order to separate the transformed cells from the non-transformed cells and, in the case of plants, to regenerate transformed plants from the transformed cells. Indeed, complete plants can be regenerated from individual transformed cells thus ensuring that all cells of the regenerated plant will contain the transgene.
In many plants, genetic transformation can be achieved by using the natural capacity of certain Agrobacterium strains to introduce a part of their Ti-plasmid, i.e. the T-DNA, into plant cells and to integrate this T-DNA into the nuclear genome of the cells. It was found that the part of the Ti-plasmid that is transferred and integrated is delineated by specific DNA sequences, the so-called left and right T-DNA border sequences and that the natural T-DNA sequences between these border sequences can be replaced by foreign DNA (European Patent Publication "EP" 116718; Deblaere et al, 1987 Meth.Enzymol. 153:277-293).
Certain plant species have proven to be recalcitrant to Aqrobacterium mediated transformation and in these species, as well as in animals, genetic transformation has been achieved by means of direct gene transfer by which DNA is inserted into the cells by physical and/or chemical means, such as by electroporation, by treatment of the cells with polyethyleneglycol (PEG), by bombardment of the cells with DNA-coated microprojectiles, etc. (WO 92/09696; Potrykus et al, 1991, Annu.Rev.Plant Physiol.Plant Mol.Biol. 42:205-225).
Genetic transformation of eucaryotic cells is generally a random event, i.e. the transgene is integrated in the genome at random positions. Often several copies (or parts of copies) of the transforming DNA are integrated in a single position, and/or at different positions, resulting in a transformed cell containing multiple copies of the transgene.
The expression of the transgene is known to be influenced by its position in the genome. For instance, a foreign DNA, when introduced in a plant cell appears to integrate randomly in the plant genome. Examination of independently transformed plants has shown a high degree of variability (up to 1 00-fold) in the expression level of the introduced gene. Several studies have shown no correlation between this "between-transformant variability" and the copy number of the introduced DNA at a given locus. It has been suggested that some of the variability in expression of introduced genes in transgenic plants is a consequence of "position effects" caused by influences of adjacent plant genomic DNA. Other factors that could contribute to the variability in expression are physiological variability of the plant material, differences in the number of independent T-DNA loci in different transformants or the inhibitory effects of certain T-DNA structures on gene expression. Between-transformant variability in expression has been observed for the majority of introduced genes in transgenic plants. The variability in expression of many introduced genes in independent transgenic plants necessitates large numbers of transgenic plants to be assayed to accurately quantitate the expression of the gene. It would be of great importance if the amount of between-transformant variability could be reduced (Dean et al, 1988, NAR 16:9267-9283).
If the transgene is under the control of a tissue-specific promoter, with the expectation that it will be expressed in selected tissues of the transformed organisms, the position effects can lead, at least in some transformants, to loss of specificity of the promoter and expression of the transgene in undesired tissues, e.g. in tissue cultured in vitro.
Factors that are known to influence the efficiency and quality of the genetic transformation process are the method of DNA delivery, specific tissue culture conditions, the physiological and metabolic state of the target cells etc. Direct gene transfer methods for instance are generally known to result in transformed organisms with a high copy number of the transgene. Many of these factors are not under the control of man.